The overall goal of this project is to examine the immunological mechanisms which may be involved in the pathogenesis of autoimmune- and infectious diseases of the central nervous system such as multiple sclerosis (MS) and chronic Lyme disease. Our studies aim at characterizing the fine specificity, function and phenotype of T lymphocytes in the above diseases. These experiments allow a better understanding of the foreign antigens that may trigger autoimmune responses in MS and also of their function with respect to cytokine secretion and chemokine receptor expression. Based on this knowledge the project attempts to develop both specific immunomodulatory treatments such as altered peptide ligands (APL) or therapies that influence immune recognition in MS in a broader way. Examples of the latter are the humanized antibody against the interleukin-2 receptor alpha chain (Zenapax) or the phosphodiesterase type IV inhibitor Rolipram. The trial with the APL peptide has been concluded, and the data published. The clinical trials with Zenapax and Rolipram are both supported through bench-to-bedside proposals. The first part of the phase II clinical trial is almost concluded and has shown that the drug is not only exceptionally well tolerated, but also highly active in inhibiting disease activity in MS. Rolipram has been recrystallized under GMP conditions, and the phase II clinical trial has started beginning of 2002. 7 patients are currently enrolled in the dose finding stage of the trial. It is too early to assess efficacy. With respect to the above clinical trials we have also begun as part of this project to characterize the mechanism of action of already approved treatments of MS, i.e. interferon-b and copolymer-1, by a combination of cDNA microarrays, quantitative PCR, ELISA, flow cytometry and other cellular immunological techniques. These experiments shall delineate the gene expression profiles and functional consequences by treatment of these drugs in vitro. As a next step, we have collected peripheral blood samples along the above clinical trials and during natural history studies of MS and examined similar parameters ex vivo in relation to the inflammatory disease activity that was documented by MRI. These studies will help us to understand better the complex mechanism of action of these compounds and eventually also the disease pathogenesis itself. Furthermore, we are developing techniques that will hopefully permit the identification of responder- and non-responder phenotypes in individual MS patients. As a further goal, we have started to examine the influence of drug combinations in vitro and hope to advance these studies in order to develop rational drug combinations. All the clinical projects are being pursued in close collaboration with the Clinical Studies Section/Office of the Chief under Henry F. McFarland, M.D. Another important project, which is currently being pursued at NIB, NINDS, NIH, addresses the question which foreign antigens, e.g. viruses or bacteria, may trigger the initiation or exacerbations of disease via a mechanism referred to as molecular mimicry. This concept refers to cross-recognition between autoantigens, e.g. derived from the myelin sheath, and antigens derived from foreign agents. For this purpose, we currently employ a novel methodology called combinatorial peptide libraries in the positional scanning format (ps-SCL) together with bioinformatic approaches to identify the entire spectrum of stimulatory ligands for autoreactive T cell clones derived from MS patients. In brief, we test T cell clones with ps-SCL, which represent highly complex mixtures of trillions of peptides, and deduce stimulatory peptide sequences from these assays before we screen the databases of all known protein sequences for potential stimulatory peptides. This technique has allowed us to identify a number of Borrelia burgdorferi peptides, the causative agent of Lyme disease, and autoantigens, for a clone that had been isolated from the cerebrospinal fluid of a patient suffering from chronic CNS Lyme disease. Thus, we could show for the first time that one can identify the target specificity of an organ-infiltrating T cell clone that had been expanded by a crude lysate of the whole bacterium. We are currently in the process of developing this methodology further and anticipate that the combination of ps-SCL and biometric data analysis will lead to advances in the identification of target antigens for autoimmune diseases, but also for tumor-specific lymphocytes or T cells that are involved in infectious disease. As an example for the latter, our data for organ-infiltrating T cells in chronic nervous system Lyme disease already suggest that the immune response in the chronic stage of the disease is directed against tissue autoantigens and that this process is thus very similar to an autoimmune disease. Currently, we also develop molecular biology strategies, i.e. the expression of cDNA clones from MS brain in a special eukaryotic expression system, for the identification of novel proteins that are expressed in MS brains and serve as targets for the autoimmune response. New projects include: The differentiation of hemopoietic stem cells into glial/neural cells. Integration of immunological studies, MRI, clinical examination, expression profiling and proteomics for the stratification of MS subtypes.